Non-alcoholic fatty liver disease (NAFLD) is prevalent (overall occurrence of ~20%) in the US adult population and is very commonly associated with obesity and diabetes (occurs in ~ 60% of diabetic patients). NAFLD presents as increased number and size of lipid droplets within the hepatocytes, and, if untreated, can progress to the inflammatory disease steatohepatitis and liver dysfunction. This proposal, in response to PAR-06-545 for the Molecular Libraries Screening Centers Network (MLSCN), proposes an assay that will enable high- throughput/high-content screening of chemical libraries to detect agents that alter hepatic lipid droplet formation. The assay employs AML-12 cells (murine hepatocyte) or HuH7 cells (derived from human hepatocytes). Cells plated in a high-throughput format, are exposed to oleic acid, which induces lipid droplet formation, fixed and stained for lipid droplets, and imaged utilizing a robotic fluorescence microscopy workstation. Automated image analysis software is utilized to quantify the number and size of lipid droplets. Our assay has an exceptionally high Z' value for reproducibility (Z' > 0.5 for the effect of oleic acid) and is inexpensive and rapid to perform. Assay efficacy was demonstrated using triacsin C, an inhibitor of long-chain acyl-CoA synthetase (ACSL), to block oleic acid-induced lipid droplet formation (Z' > 0.5 for this effect). Thus, the assay will provide an excellent method to identify new agents that could be developed for treatment of NAFLD disease or used as chemical probes to identify proteins in the hepatic triglyceride ester synthesis and lipid droplet formation pathways. [unreadable] [unreadable] [unreadable]